LOCUS       sequence-349904-        6426 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of recombinant fusion protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6426)
  AUTHORS   Sowa ST, Galera-Prat A, Wazir S, Alanen HI, Maksimainen MM, Lehtio L
  TITLE     A molecular toolbox for ADP-ribosyl binding proteins.
  JOURNAL   Cell Rep Methods. 2021 Dec 20;1(8):100121. doi: 
            10.1016/j.crmeth.2021.100121. Epub 2021 Nov 11.
  PUBMED    34786571
REFERENCE   2  (bases 1 to 6426)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Rep 
            Methods."; date: "2021-12-20"; volume: "1(8):100121. doi"; pages: " 
            10.1016/j.crmeth.2021.100121. Epub 2021 Nov 11"
COMMENT     Sequence Label: Gi-c001
FEATURES             Location/Qualifiers
     source          1..6426
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             4..21
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             46..66
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1203..1220
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1273..1291)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1287..1334
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1371..1826
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1458..1477)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1668..1689
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1918..2733)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1918..2733)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2641..2660
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2725..2744
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2855..3443
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3344..3363
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3597..3614
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3629..3768
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3694..3713)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3854..3876
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3870..4061)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4870..5952)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5914..5933
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5953..6030)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6236..6255
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6339..6358
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6339..6357
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6358..6382
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             6397..6419
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgcaccatc atcatcatca ttcttctggt gtagatctgg gtaccgagaa cctgtacttc
       61 caatccatgg gttgcaccct gagcgcggaa gacaaggcgg cggtggagcg tagcaaaatg
      121 atcgaccgta acctgcgtga agatggcgaa aaggcggcgc gtgaagtgaa gctgctgctg
      181 ctgggtgcgg gtgaaagcgg caagagcacc attgttaagc agatgaaaat cattcacgaa
      241 gcgggctaca gcgaggaaga gtgcaagcag tacaaagcgg tggtttatag caacaccatc
      301 caaagcatca ttgcgatcat tcgtgcgatg ggtcgtctga aaattgactt tggtgatagc
      361 gcgcgtgcgg atgatgcgcg tcaactgttc gttctggcgg gtgcggcgga agagggcttt
      421 atgaccgcgg aactggcggg tgtgatcaag cgtctgtgga aagacagcgg cgttcaggcg
      481 tgctttaacc gtagccgtga gtaccaactg aacgatagcg cggcgtacta tctgaacgac
      541 ctggatcgta tcgcgcagcc gaactatatt ccgacccagc aagacgtgct gcgtacccgt
      601 gttaagacca ccggtatcgt ggaaacccac ttcaccttta aggacctgca cttcaaaatg
      661 tttgatgttg gtggccaacg tagcgagcgt aagaaatgga ttcactgctt cgagggcgtg
      721 accgcgatca tcttctgcgt tgcgctgagc gactacgatc tggtgctggc ggaagacgaa
      781 gagatgaacc gtatgcacga gagcatgaag ctgttcgaca gcatctgcaa caacaaatgg
      841 tttaccgata ccagcatcat tctgttcctg aacaagaagg acctgttcga agagaaaatc
      901 aagaaaagcc cgctgaccat ttgctacccg gagtatgcgg gtagcaacac ctacgaagag
      961 gcggcggcgt atatccagtg ccaattcgaa gacctgaaca agcgtaaaga taccaaggag
     1021 atttataccc actttacctg cgcgaccgac accaaaaacg tgcagttcgt ttttgacgcg
     1081 gtgaccgatg ttatcatcaa gaacaacctg aaggattgcg gcctgttcta agacggtctc
     1141 cagtaaaggt ggatacggat ccgaattcga gctccgtcga caagcttgcg gccgcactcg
     1201 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     1261 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     1321 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     1381 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     1441 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     1501 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     1561 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     1621 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     1681 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     1741 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     1801 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     1861 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     1921 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     1981 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     2041 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     2101 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     2161 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     2221 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     2281 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     2341 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     2401 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     2461 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     2521 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     2581 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     2641 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     2701 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     2761 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     2821 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     2881 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     2941 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3001 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3061 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3121 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3181 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3241 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3301 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     3361 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3421 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3481 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3541 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3601 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     3661 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3721 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     3781 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3841 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     3901 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     3961 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     4021 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     4081 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     4141 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     4201 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     4261 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     4321 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     4381 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     4441 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     4501 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     4561 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     4621 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     4681 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     4741 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     4801 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     4861 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4921 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     4981 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     5041 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     5101 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     5161 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     5221 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     5281 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     5341 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     5401 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     5461 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     5521 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     5581 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     5641 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     5701 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     5761 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     5821 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     5881 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     5941 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     6001 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     6061 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     6121 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     6181 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     6241 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     6301 gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga
     6361 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat
     6421 atacat
//
